Characterization of New Punch Mutations: Identification of Two Additional Mutant Classes

The Punch locus of Drosophila melanogaster which encodes the pteridine biosynthetic enzyme, GTP cyclohydrolase, is genetically complex. Lethal alleles of the locus resolve into an array of interallelic complementation groups, and at least one class of mutations is developmentally specific, affecting GTP cyclohydrolase activity only in the heads of adults. All previously isolated Punch alleles were identified on the basis of a mutant eye color phenotype. By screening mutagenized chromosomes over Punch region deficiencies, we have now isolated new alleles on the basis of lethal and visible phenotypes. Most of these alleles fall into previously identified genetic classes, but two new classes of mutations were also found. One class contains two alleles that behave as dominant lethal mutations in some genetic backgrounds. The other class represents a second developmentally specific set of alleles that affect the function of the Punch locus only during embryogenesis.     

Suppression of postprandial glucose, insulin, C-peptide, and glucose-dependent insulinotropic peptide (GIP) in man by oral administration of a prostaglandin analogue (enprostil)

Enprostil, a long-acting, orally active dehydroprostaglandin E2 with cytoprotective and gastric antisecretory properties, is a potent inhibitor of meal-stimulated gastrin release. Recent data have suggested suppression of additional other gastrointestinal peptide hormones following single doses of enprostil. The current investigation was conducted to further clarify the effects of enprostil administration on gastrointestinal hormones and glucose metabolism under physiologic conditions and to determine whether these effects were present following multiple doses of the agent. Enprostil 70 mcg/d and its placebo were each administered for 7 1/2 days to eight normal male subjects in a study of crossover design, each treatment period lasting 7 1/2 days and separated by a 7 day washout period. Subjects received a test meal on days 1 and 8 and an oral glucose challenge on day 3 of each treatment period following enprostil or its placebo. Following the test meal, there was a delay and suppression of the maximum measured serum glucose levels. Mean overall peak glucose concentrations were lower during the enprostil phase compared to placebo (112 vs. 121 mg/dd, P = 0.025) with a trend toward delay in the time to achievement of peak glucose concentrations. Mean overall peak levels for insulin, C-peptide, and glucose-dependent insulinotropic peptide (GIP) were significantly suppressed by 36%, 16% and 60%, respectively by enprostil when compared to placebo. The overall integrated postprandial responses for insulin, C-peptide, and GIP were significantly reduced by 42%, 39% and 90%, respectively while that for glucose above baseline was reduced by 44% (P = 0.098). Similar effects were present following the oral glucose challenge.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical and physical characterization of rabbit sperm acrosome stabilizing factor

Rabbit Acrosome Stabilizing Factor (ASF) is an epididymal product that reversibly inhibits the process of sperm capacitation. The native molecular weights of the monomer and dimer ASF were determined from sedimentation and diffusion data at 129,000 and 259,000 Mr. The monomer is composed of 92,000 and 38,000 Mr subunits according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with size heterogeneity demonstrated for the latter. The stoichiometry of the subunits appears to be one-to-one by gel scanning. Amino acid and carbohydrate compositions are characteristic of a globular glycoprotein, which is high in cysteine content and is 8.3% carbohydrate by weight. The sugar composition suggests the presence of both high mannose and complex N-linked oligosaccharides with the unusual feature of appreciable amounts of glucose. The isoelectric character of ASF spans a range from 5.3 to 7.0.     

Experimental manipulations of the phytoplankton periodicity in large limnetic enclosures in Blelham Tarn,English Lake District

This paper reviews the results of experimental manipulations, carried out during the period 1977–1983, on the phytoplankton maintained in the limnetic enclosures at Blelham Tarn, English Lake District. Three categories of manipulations are considered.The effects of variation in the scale and frequency of phosphorus loading (range: 0.3 to 2.5 g P m^–2 a^–1) upon the mean phytoplankton biomass, its seasonal distribution and specific dominance are shown to conform to well-established patterns and relationships observed in natural lakes. Much of the seasonal variability in species dominance occurred independently of nutrient ratios, though carbon availability has been critical at times. Attempts to manipulate the rates of removal of phytoplankton by grazing have confirmed that they act selectively against certain smaller species only, that they alter the rate of successional change, rather than its direction, and that they have little lasting influence upon the total phytoplankton standing crop. Attempts to manipulate rates of sinking loss through artificial enlargement of the epilimnetic circulation also regulated the light-conditions experienced by suspended phytoplankton.Growth-rate relationships to an index of light exposure and to temperature fluctuation are also derived for several species and are related to morphological and physiological characters of the organisms concerned. These interpretations are briefly reviewed in relation to periodic cycles in natural lakes.     

DNA sequence specificity of the pyrrolo[1,4]benzodiazepine antitumor antibiotics. Methidiumpropyl-EDTA-iron(II) footprinting analysis of DNA binding sites for anthramycin and related drugs

Anthramycin, tomaymycin, and sibiromycin are members of the pyrrolo[1,4]benzodiazepine [P(1,4)B] antitumor antibiotic group. These drugs bind covalently through N2 of guanine and lie within the minor groove of DNA [Petrusek, R. L., Anderson, G. L., Garner, T. F., Fannin, Q. L., Kaplan, D. J., Zimmer, S. G., & Hurley, L. H. (1981) Biochemistry 20, 1111-1119]. The DNA sequence specificity of the P(1,4)B antibiotics has been determined by a footprinting method using methidiumpropyl-EDTA-iron(II) [MPE.Fe(II)], and the results show that each of the drugs has a two to three base pair sequence specificity that includes the covalently modified guanine residue. While 5'PuGPu is the most preferred binding sequence for the P(1,4)Bs, 5'PyGPy is the least preferred sequence. Footprinting analysis by MPE.Fe(II) reveals a minimum of a three to four base pair footprint size for each of the drugs on DNA with a larger than expected offset (two to three base pairs) on opposite strands to that observed in previous analyses of noncovalently bound small molecules. There is an extremely large enhancement of MPE.Fe(II) cleavage between drug binding sites in AT rich regions, probably indicating a drug-induced change in the conformational features of DNA which encourages interaction with MPE.Fe(II). In the presence of sibiromycin or tomaymycin the normally guanine-specific methylene blue reaction used in Maxam and Gilbert sequencing cleaves at other bases in defined positions relative to the drug binding sites. Finally, modeling studies are used to rationalize the differences and similarities in sequence specificities between the various drugs in the P(1,4)B group and their reactions with DNA.     

Repair of closely opposed cyclobutyl pyrimidine dimers in UV-sensitive human diploid fibroblasts

An enzyme-sensitive site assay has been used to examine the fate of closely opposed pyrimidine dimers (bifilar enzyme-sensitive sites) in fibroblasts from individuals afflicted with various genetic disorders that confer increased cellular sensitivity to UV radiation. The disappearance of bifilar enzyme-sensitive sites was found to be normal in cells from individuals with Fanconi's anemia, Cockayne's syndrome, dyskeratosis congenita and the variant form of xeroderma pigmentosum. The rate of bifilar enzyme-sensitive site removal in XP cells assigned to complementation group C was reduced by an amount similar to that observed for the repair of isolated dimers. Our results indicate that the initiation of repair at closely opposed dimers is slow in XP-C cells but normal in all other cells examined.     

Blood–Brain Transfer of Glucose and Glucose Analogs in Newborn Rats

Little is known of the selectivity of the blood-brain barrier at birth. Hexoses are transported through the barrier by a facilitating mechanism. To study the capacity of this mechanism to distinguish between analogs of D-glucose, we compared the transport of fluorodeoxyglucose, deoxyglucose, glucose, methylglucose, mannose, galactose, mannitol, and iodoantipyrine across the cerebral capillary endothelium in newborn Wistar rats. Cerebral blood flow, glucose consumption, and the blood-brain permeabilities of the hexoses were 25-50% of the adult values but the ratios between the permeabilities of the individual hexoses were similar to the ratios observed in adult rats. The mannitol clearance into brain was considerably higher than in adult rats (about 10-fold), indicating a higher endothelial permeability to small polar nonelectrolytes. The brain water content was higher in newborn than in adult rats and was associated with a higher steady-state distribution of labeled methylglucose between brain and blood. Hexose concentrations were determined relative to whole blood because the apparent erythrocyte membrane permeability to glucose was as high as in humans and thus considerably higher than in adult rats. The half-saturation concentration of glucose transport across the blood-brain barrier was considerably higher than in adult rats, about three-fold, suggesting that net blood-brain glucose transfer is less sensitive to blood glucose fluctuation in newborn than in adult rats.     

Oesophageal peristalsis in the cat: the role of central innervation assessed by transient vagal blockade

Studies were performed on five cats to assess the role of extrinsic vagal innervation in the control of peristalsis in the smooth muscle oesophagus. Transient vagal nerve blockade was accomplished by cooling the cervical vagosympathetic nerve trunks previously isolated in skin loops on each side of the neck. Peristalsis throughout the body of the oesophagus was monitored using a continuously perfused multilumen manometry tube. Striated and smooth muscle portions of the esophagus were delineated by abolishing smooth muscle activity with atropine. Secondary peristalsis was assessed by intra-oesophageal balloon distension studies. The threshold volume for balloon-induced secondary peristalsis was lower in the smooth muscle oesophagus. Unilateral vagal blockade reduced the incidence of primary and secondary peristalsis in the striated muscle oesophagus but not in the smooth muscle oesophagus. Bilateral vagal nerve blockade abolished primary swallow-induced peristalsis and secondary peristalsis in both the smooth and striated muscle cat oesophagus. Administration of cholinergic agents or adrenergic blocking agents failed to restore secondary peristalsis in the smooth muscle oesophagus during vagal cooling. We conclude that connections to the central nervous system via the vagal nerve trunks are required for normal secondary as well as primary peristalsis in both the smooth and striated muscle portions of the cat oesophagus.     

Purification and characterization of a bone metalloproteinase that degrades gelatin and types IV and V collagen

A third metalloendopeptidase activity, gelatinase, has been completely separated from the collagenase and proteoglycanase activities of rabbit bone culture medium. Although the proteinase could not be purified to homogeneity in large amounts, it was possible to obtain accurate molecular weight values and activity after electrophoresis on non-reduced SDS/polyacrylamide gels. The latent form had an Mr of 65 000 which could be activated with 4-aminophenylmercuric acetate, APMA, to a form of Mr 61 000; under reducing conditions the latent and active forms had Mr of 72 000 and 65 000, respectively. Trypsin was a very poor activator of the latent enzyme. Gelatinase degraded gelatins derived from the interstitial collagens and it also had low activity on native types IV and V collagen and on insoluble elastin. Gelatinase acted synergistically with collagenase in degrading insoluble interstitial collagen. The specific mammalian tissue inhibitor of metalloproteinases inhibited gelatinase by forming a stable inactive complex. Comparison of the properties of gelatinase with those of collagenase and proteoglycanase suggest that the three proteinases form a family which together are capable of degrading all the major macromolecules of connective tissue matrices.